Isolation of Piperine from Black Pepper William W. Epstein
University of Utah, Salt Lake City, UT 84112
David F. ~ e t r l and Jimmy L. seide12 University of
Wisconsin-Oshkosh, Oshkosh, WI 54901
The isolation of natural products from readily available Black
pepper is known to have a variety of physiological spices has
played an important role in exposing students properties including
~arminative,~ diaphoretic,' diuretic," to the laboratory practices
of organic chemistry. For exam- li~olviic (111.~ and insecticidal
(12-1.5, ~ r o ~ e r t i e s , in addi- ple, the isolation of
trimyristin ( I ) , carvone (21, eugenol(3), and anethole (4) have
all been described and introduce the beginning organic chemistry
student to a variety of tech- niques. We wish to add a new and
interesting member to this list. Described below is the isolation
of the alkaloid piperine (figure) from black pepper.3 The procedure
allows students to be introduced to the use of a reflux apparatus
and the techniques of trituration, recrystallization, suction
filtration, thin-layer chromatography, and melting point
determination using either semimicro- or microscale labware and
techniques. The experiment can be completed in a single 3-4 h
laboratory period, and as such, is an ex- cellent first experiment
for beginning organic chemistry students.
Historical Background Commerciallv available black Deuuer is the
dried. full-
grown but ungpe h i t of the pe;er&l woody vine Piper nigrum
L., while white pepper is obtained from the dried ripe fruit by
removing the outer portion of the pericarp after soaking in water.
Both black and white pepper find their way into homes as flavoring
agents and may be bought in many forms such as crude, whole or
ground pep- per fruits, as an oil,' and as an oleore~in.~ All forms
of pep- per are generally regarded as safe in FDA regulatory sta-
tus with the highest average use level reported in nut products
(0.42% white pepper) and in baked goods (0.2% oleoresins) (10).
tion& its widely known activity on the taste buds, which
produces a reflex increase in gastric secretion. Folk medi- cine
effects of pepper include its use as a carminative, stimulant and
tonic, as well as for treatment of various cancers (16). In
addition to these uses (lo), there are Chi- nese folklore reoorts
of white ueuuer involved in the treat-
A .A
ment of malaria, stomachache, and cholera.1 According to Dewein
(17), the pungency of black pepper
was reported by Oerstedt (18) in 1821 to be due to the pres-
ence of piperine (figure), the structure of which was estab-
'American Chemical Societv Petroleum Research Fund Scholar.
2Present address: EPA-NEIC, Building 53, Box 25227, Denver
Federal Center, Denver, CO 80225. "resented at the 12Ih Biennial
Conference on Chemical Educa-
tion. Davis, CA, August 1-7, 1992. 4 P e ~ ~ e r oil is the
volatile oil obtained by steam distillation of black
pepper' and contains a variety of mono: and sesquiterpenes, but
none of the pungent agents (5-9).
'Black pepper oleoresin is obtained by solvent extraction of the
crude pepper followed by removal of the solvent and contains the
pungent principles as well as the volatile oil in pepper (10).
%arminative: Anything used as a remedy for flatulence (an accu-
mulation of gas in the intestinal tract).
'~iaphoretic: Any ingested substance that induces perspiration.
8Diuretic: An agent that increases the excretion of urine.
gLipolytic: Capable of breaking down and decomposing fat. "Cholera:
An acute bacterial disease that principally causes seri-
ous intestinal disorders.
Piperine Kperidine Kperettine
Piperanine
Piperine and related alkaloids found in black pepper.
598 Journal of Chemical Education
lished by Ladenburg and Scholtz in 1894 (19). Since then, it has
been determined that black pepper contains approx- imately 2-4%
volatile oils and 5-9% of the alkaloids piper- ine, pipendine,
piperethe, as well as several other minor components, most notably
piperanine and pipercide (fig- ure) (12, 20-25). Piperine and
piperanine are the known pungent agents (24, 251, while pipercide
is one of three known insecticidal agents present in black pepper
(12.15). White pepper has thk sake pungent principie-s and Aka-
loids as black pepper but contains only small amounts of the
volatile oils. Because piperine can be isolated only in 240byirld
from blark pepper and is valuable as a pungent aeent. several
svnthetic routes have been devisedi26). - . . .
Classically, ppperine is isolated by ethanol extraction of mound
DeDoer followed bv an overnieht ~recinitation of -
piperinefrbk 10% alcohol' potassiun-hydroxi~e (27). Be- cause of
the relatively short time available during most laboratory periods,
an alternate method involving the ex- traction of ~ i ~ e r i n e
into dichloromethane and its ~recioita-
. .
tion via trii&ation of the crude oils with diethyl ether was
developed.
~xperimental" Extraction
Add 5.0 g of pure ground pepper1' and 10 mL of dichloromethane
to a 50-mL round-bottomed flask. Use the round-bottomed flask as
the basis for a reflux appara- tus having a water-cooled condenser
and heating mantle. Heat the sample to reflux, and then maintain a
gentle re- flux for 20 min. ARer the required reflux period, lower
the heating mantle and allow the reflux apparatus to cool for 5
min. Suction filter the slurry with the aid of a 4.5-cm Biichner
funnel, washing the pepper grounds once with 5 mL of
dichloromethane. Remove two or three drops of the extract and place
it in a capped vial for use in the thin- layer chromatographic
analysis.
Transfer the extract obtained above to a clean 25-mL
round-bottomed flask and concentrate in ~acuo. '~ The re- sulting
olive-brown, viscous oil should be cooled in an ice-
"The experimental procedure can be adapted for use with
microscale glassware after the extraction has been completed.
"We have found that the use of inexpensive grades, and certain
national brands, of ground black pepper do not yield isolable
amounts of piperine when used. However, we have found that the
Schilling brand of pureground black pepper marketed by McCormick
& Co., Inc. gives cansistent and reliable results.
l 3 The use of a rotary evaporator is ideal. However, any method
may be utilized as long as all the solvent is removed. Any
remaining traces of CH2C12 will prevent the precipitation of
piperine upon tritu- ration with Ft-O~ . -. . . . . . . . .. . -
.
14This pro&&re assists in removing the last traces of
CH2Cl, that may be present and that prevent complete precipitation
of the ex-
. .
tracted piperine. ' = I f a copious amount of precipitate is not
present, wncentrate the
sample in vacuoagain and repeat thetrituration. Generally,
astudent who failed to obtain a precipitate the first time should
now obtain an appropriate amount of material. If not, concentrate
and triturate as needed to precipitate the product.
16Typically, yields of approximately 2% or 100 mg are ob-
tninea
17Literature melting point 130-132.5 OC: Weast, R. c., ~ d . CRC
Handbook of Chemistry and Physics. 5ern ed.; CRC Press: West Palm
Beach. F L 1978~
- -.-.
"Alor ch Chem cal Company ot Mi waukee, W . sells piperme in 5 g
and 25 g lots. Tne mater a is of s~f l clent purity (97%) lor
preparing TLC slandaras an0 as a reference stanoaro for mixed
meltma w nts
- . determinations (mp 131-134C). '9~ieselgel 60 F, plates
having a 0.2 mm coating thickness are
recommended.
bath and then 3 mL of cold ether added to the oil while gently
stirring for 3 4 min. Some piperine may precipitate .at this point,
but remove the solvent in uacuo anyway. Once again cool the
resulting oil in an icebath and then add 3 mL of cold ether to the
oil while gently stirring to promote the precipitation of
piperine.14 Allow the flask to cool for an additional 10 min with
occasional ~tirring.'~ Iso- late the straw-yellow crystals of crude
piperine by suction filtration with the aid of a 1.5-cm Hirsch
funnel. Wash the crystals twice with 2-mL portions of cold ether.
Place a small portion of the filtrate in a capped vial for use in
the thin-layer chromatographic analysis.
Recrystallization Place the crude piperine isolated above into a
13 x 100
mm test tube and dissolve it in a minimum amount of hot 3:2
acetone:hexane solution. Once all the solid has dis- solved, allow
the test tube to sit undisturbed for 15 min at room temperature.
Rod-like, yellow crystals of piperine should be present. Cool the
solution for an additional 30 min in an ice-bath before isolating
the purified piperine by suction filtration with the help of a
1.5-cm Hirsch funnel. Wash the crystals once with a 2-mL portion of
cold ether, allow them to air dry for several minutes, and then
obtain the weight of the crystal^.'^ The melting pointL7 of the pu-
rified piperine now can he determined and the identity of the
product confirmed by mixed melting point, thin-layer
chromatographic analysis, or spectral analyses.'8
Thin-Layer Chromatogra~hic Analvsis - .
Transfer a small portion of the purified piperine crystals to a
small vial and dissolve them in a drop or two of ace- tone. Prepare
a silica-gel thin-layer chromatography plate" for the spotting of
four samples. With the aid of a capillary, place a sample of the
crude oil remaining from the extraction procedure, a sample of the
filtrate from the triturationlisolation procedure, the sample
prepared from the purified piperine, and a sample of a piperine
standard on separate points of the thin-layer plate. Develop the
plate using 3:2 acetone:hexane. Visualize under UV illumination and
stain in an iodine chamber.
Acknowledgment Acknowledgment is made to the Donors of The
Petro-
leum Research Fund, administered by the American Chemical
Society, for support of this research.
Literature Cited I Prmk. P . Rabrhl.T.Snel1.J Ydtrr (:
.~'lllrnr.J J Chrm E,lh 1971.18.255 2 hlum\.S L . Plrk*.nng. \I J
('hem E d ~ c I978 ill. :l 3 Nlnmdn M 5 Iln*oanalhA J Chenr N u .
1976.57 2G3. I (innn. l l I . J (hem E d u 1980.57, l.